O-linked ?-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification in mammals, decorating thousands of nuclear, cytoplasmic and mitochondrial proteins. O-GlcNAc cycling is an essential regulator of cell metabolism, cell cycle progression and cell death, and is dysregulated in numerous human diseases, such as cancer, diabetes and cardiac arrhythmia. Despite its broad pathophysiological significance, major aspects of O-GlcNAc signaling remain obscure, including how O-GlcNAc transduces biological information inside the cell. Recently, several studies showed that O-GlcNAcylation induces protein-protein interactions in processes as diverse as chromatin remodeling, deubiquitination and nuclear envelope assembly, suggesting that O-GlcNAc may signal through conserved modes of protein-protein interaction. However, little is known about either the structure or function of these intracellular glycoprotein-protein complexes. We hypothesized that mammalian reader proteins might exist and recognize O-GlcNAc moieties for signaling purposes. By analogy to reader proteins for other post-translational modifications, we reasoned that dedicated O-GlcNAc readers - or protein domains common to many readers - might relay glycosylation-encoded signals. In preliminary studies, we used a new biochemical approach to identify several proteins that bind specifically and directly to O-GlcNAcylated (but not unglycosylated) peptides and proteins in vitro and in cells. Interestingly, one group of putative O-GlcNAc reader proteins also binds to phosphoproteins, suggesting that they may be signal integrators, mediating the previously described crosstalk between O-GlcNAc and O- phosphate. This result may have wide-ranging implications for our understanding of cell signaling through post- translational modifications. The objective of this project is to characterize the biochemical and cell biological functions of the O- GlcNAc reader proteins that we identified. We will accomplish this goal through three specific aims. In Aim 1, we will define the biochemical scope of O-GlcNAc binding by the candidate readers. In Aim 2, we will identify the endogenous glycoprotein binding partners of O-GlcNAc reader proteins, and establish the role of these interactions in cell signaling. In Aim 3, we will determine the biophysical basis of an O-GlcNAc-mediated protein- protein interaction by solving the crystal structure of a reader protein bound to a glycosylated ligand. Our work will significantly advance the field of cell signaling by characterizing O-GlcNAc reader proteins at the cell biological, biochemical and atomic levels.